This project proposes to study the biochemical and molecular mechanisms involved in the regulation of apoptosis, with a special emphasis on amplifying bystander-mediated apoptosis for treatment of cancer. The long-term objectives of this project focus on elucidation of mechanisms by which adenovirus-mediated FasL expression confers programmed cell death (PCD) and generates apoptotic vesicles that function to cause additional PCD in adjacent cells (bystander effect). Specific emphasis will be devoted to the role of sphingolipids, and their effects on alternative splicing and proteasome function in this process. We will test the hypothesis that agents that perturb sphingolipid metabolism sensitizes uninfected and/or resistant tumor cells to promote a more proapoptotic phenotype, and facilitates bystander activity and achieve greater tumor cell killing in tumors treated with AdGFPFasL virus. The following three specific aims will address this issue. Specific Aim 1: Will agents that increase ceramide levels generate a more proapoptotic phenotype resulting in increased bystander activity in AdGFPFasL-treated prostate tumors and by what mechanism? Specific Aim 2: Determine the mechanisms of resistance to P1 in vitro and assess if it can be modified by antisense, siRNA, chemotherapy and ceramidase inhibitors to facilitate bystander activity. Specific Aim 3: Determine if we can modify the levels of anti-apoptotic molecules in vivo and will this amplify the bystander effect elicited by AdGFPFasL. Important basic and pre-clinical information to arise from these studies will be the understanding of mechanisms that mediate the bystander effect and its contribution to prostate cancer PCD in vitro and in vivo.